INTRODUCTION

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Tethering interactions between the cytoplasmic domains of integral membrane and other proteins, mediated by ankyrin or proteins containing ankyrin-like repeat structures, play fundamental roles in diverse biological activities including growth and development (1-5), intracellular protein trafficking (6-8), the establishment and maintenance of cellular polarity (9-12), cell adhesion (13, 14), signal transduction (2, 15-19), and mRNA transcription (20, 21). Ankyrin, including its many isoforms (reviewed in Refs. 22 and 23), is also the most ubiquitous adapter protein mediating linkage of membrane proteins with the spectrin-based skeleton, both at the plasma membrane (reviewed in Refs. 23-25) as well as with internal membrane compartments including the Golgi apparatus (6-8). A characteristic feature of ankyrin is the presence of well conserved 33-residue repetitive units (ankyrin repeats) that individually or in combination bind to transmembrane proteins (reviewed in Ref. 23). Because no data on the structure of an ankyrin-binding domain in an integral membrane protein exist, it is unclear how so many diverse membrane proteins can bind specifically to a single ankyrin molecule (26, 27).

The -subunit of Na,K-ATPase interacts with ankyrin (11, 28). This interaction stabilizes Na,K-ATPase at the plasma membrane and enables its transport from the endoplasmic reticulum to the Golgi (8). Cytoplasmic domains (CD)¹ II and III of -Na,K-ATPase bind ankyrin in vitro. These sequences appear to act independently, and those in CDII (residues 140-290) account for most of the affinity of -Na,K-ATPase for ankyrin (28, 29). We now identify a 25-residue minimal ankyrin-binding (MAB) sequence within CDII of -Na,K-ATPase and report its three-dimensional structure. This peptide forms a loop-on-a-stalk motif well suited for interaction with one or more of the putative -hairpin tips of the ankyrin repetitive unit (5). We anticipate that the ankyrin-binding structure identified here will emerge as a common feature of other ankyrin-binding proteins. Portions of this work have been presented in abstract (30).

	MATERIALS AND METHODS

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Preparation and Purification of Recombinant Peptides-- Oligonucleotides were designed to amplify constructs IIa, IIb, and IIc from a construct encoding CDII of -Na,K-ATPase (28). Amplification products were subcloned into TA vectors (Invitrogen) and sequenced by the dideoxynucleotide chain termination method (U. S. Biochemical Corp.). Constructs were expressed as fusions with Schistosoma japonicum glutathione S-transferase (SjGST) using the pGEX-2T prokaryotic expression vector (Amersham Pharmacia Biotech) and purified using glutathione-Sepharose (28). SjGST was expressed as a control peptide. All peptides were eluted with 50 mM Tris-HCl, 5 mM reduced glutathione, pH 8.0, and dialyzed into ankyrin-binding buffer (ABB; 50 mM Tris-HCl, pH 6.9, 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 1 mM EGTA, 1 µM phenylmethylsulfonyl fluoride, 1 mM Pefabloc SC). Proteins were analyzed by SDS-PAGE.

Ankyrin Binding Assay-- Each fusion protein (50 µg at 1 µg/ml) was conjugated to 50 µl of a 50% slurry of glutathione-agarose for 1 h at 4 °C with gentle rotation. Ankyrin (Ank_R) was purified by extraction of spectrin-depleted fresh erythrocyte inside-out vesicles with 1 M KCl, followed by ion exchange chromatography on DEAE cellulose (31). Ank_R binding was assayed by adding 25 µg to the peptide-conjugated beads in a total volume of 500 µl in ABB, overnight incubation at 4 °C, and analysis of the bead fraction by SDS-PAGE. Ankyrin was detected by Western blotting with specific antibodies (28). Confluent MDCK cells extracted in situ were used to prepare a high salt extractable cytoskeletal fraction (Fx2) enriched in MDCK cell ankyrin (Ank_G) (28). Conjugated beads were incubated with Fx2 (300 µg of total protein), and bound ankyrin was detected as above. Other procedures and antibodies were as before (7, 28).

Structure Determination-- Fusion peptide IIa was purified by high pressure liquid chromatography gel filtration into 50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 2 mM -mercaptoethanol. Crystals were prepared at room temperature by hanging drop vapor diffusion against a reservoir of 30% polyethylene glycol 4000, 100 mM bis-tris-propane, 150 mM NaCl, 40 µM -mercaptoethanol, pH 8.8 (32). Diffraction data were collected with a R-AXISII imaging plate detector mounted on a Rigaku 200HM generator at 170 °C using a crystal flash frozen in crystallization buffer. Data were processed using the program DENZO and SCALEPACK (33) and were 86% complete (>2). The structure was determined by molecular replacement using the published crystal structure of GST from S. japonica (34) and the program AMORE (35) of the CCP4 program suite (33). Successive rounds of model building and simulated annealing refinement were carried out with the program X-PLOR version 3.851 (36) and the CCP4 program suite. The graphic display program O, version 5.10 (37) was used to build and correct the structure manually. Each residue of the final model was checked by the omit map.

	RESULTS AND DISCUSSION

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The Minimal Ankyrin-binding Domain of -Na,K-ATPase-- Recombinant peptides derived from CDII of rat -Na,K-ATPase (28) were prepared as fusion proteins with GST and assayed for their ability to bind Ank_R (from red cells) or Ank_G (from Madin-Darby canine kidney cells) (Fig. 1). All peptides were of the predicted molecular mass, soluble, and readily purified (Fig. 1B). Peptide IIa (residues 142-166 of -Na,K-ATPase numbered according to Ref. 38) retained full binding activity to both Ank_R and Ank_G and constituted the MAB sequence. CDII peptides not encompassing MAB, such as peptide IIc (residues 168-286), were devoid of activity.

View larger version (30K): [in this window] [in a new window]   Fig. 1.   Identification of the MAB domain of -Na,K-ATPase. A, schematic representation of the five cytoplasmic domains of -Na,K-ATPase and their relationship to the ankyrin-binding peptide sequences identified here. Codon positions defining each peptide are shown. B, each depicted peptide (II-IIC) was prepared as a fusion construct with SjGST and examined for its ability to bind at various concentrations either purified AnkR (ANK1, from human red cells) or kidney ankyrin AnkG (ANK3) derived from whole MDCK cell lysates. The top panel shows Coomassie Blue-stained SDS-PAGE analysis of each peptide, as well as the entire MDCK cells extract applied to the affinity column to detect AnkG (ANK3) binding. To detect AnkR (ANK1) binding, purified erythrocyte ankyrin was used. Peptide IIA, sequence SYYQEAKSSKIMESFKNMVPQQALV, represents the minimal active sequence detected.

GST-MAB Binds Ankyrin with High Affinity-- Prior studies have detected Na,K-ATPase affinities for ankyrin in the range of 50-2600 nM (11, 39, 40). Of interest was whether GST-MAB bound ankyrin with comparable affinity. Purified ankyrin (Ank_R) was biotinylated, and its binding to GST-MAB or GST was measured (Fig. 2). Nonlinear regression analysis based on a bi-molecular binding model indicated that ankyrin specifically bound GST-MAB with a K_d = 118 ± 50 nM, in agreement with the affinity of intact Na,K-ATPase for ankyrin. Thus, although other regions of -Na,K-ATPase such as the ALLK motif identified in CD3 may contribute to its interaction with ankyrin (28, 29), the 25 residues identified here are sufficient and bind specifically to ankyrin but not to other proteins present in the whole kidney lysates (Fig. 1). GST alone was devoid of significant binding activity (Fig. 2). In separate studies we have also demonstrated that loss of these 25 residues in -Na,K-ATPase abrogates its binding to ankyrin in vivo (41).

View larger version (32K): [in this window] [in a new window]   Fig. 2.   GST-MAB binds ankyrin with high affinity. Top, purified AnkR was biotinylated and incubated with GST-MAB (peptide IIa) or GST. Lane A, purified biotinylated ankyrin (30 ng); lanes 1-8, respectively, 0.001, 0.005, 0.01, 0.05, 0.1, 0.3, 0.5, and 1.0 µM ankyrin. Shown are the autofluorograms of the bound fractions for either GST-MAB or GST alone. Bottom, the ankyrin in the GST-MAB () or GST () bound fractions as a function of the ankyrin concentration. Regression analysis yielded an estimated Kd = 118 ± 50 nM and a Bmax = 6.1 ± 0.7 × 105 (arbitrary units).

Structure Determination of MAB-- Attempts to crystallize -Na,K-ATPase CDII or MAB alone were not successful. Because the GST-MAB fusion peptide contained an ankyrin-binding domain that was fully active, carrier-mediated crystallization was used to obtain structural information (32). The active GST fusion peptide containing MAB (construct IIA, Fig. 1, and Fig. 3) yielded ordered crystals (space group of P4(3)2(1)2, with a = b = 92.17 and c = 57.57). Diffraction of this crystal at 170 °C yielded a data set that was 86% complete (>2) and allowed the determination of the complete structure of the GST-MAB fusion peptide with an R-factor of 19.8 and an R_free of 36.5 (1% of total data) at 2.6 Å resolution (Fig. 3), using a molecular replacement strategy based on the known crystal structure of S. japonica GST (SjGST) (which has one molecule in the asymmetric unit and 41% solvent) (34). The refined model displayed a continuous main chain electron density and consisted of two domains, one representing SjGST and the other MAB (Fig. 3). Each residue of MAB was verified with an electron density omit map. Both the GST and MAB domains were well defined when the total structure was checked with the 2F_o  F_c map, including the side chains of Leu¹¹⁸, His²¹⁵, and Lys²¹⁸, residues disordered in the published structure of SjGST (34). Backbone residues 1-210 of SjGST superimposed on those of SjGST-MAB with a RMS deviation of 1.02 Å. Differences between SjGST and SjGST-MAB were found in the position of Met¹ and the positions of residues 211-218, which flank a region of disorder in SjGST (but not in SjGST-MAB). There were no discernible hydrophobic or salt interactions between GST and MAB. A single H-bond was detected between Arg²²⁴ (residue 4 of the linker sequence) and Gln²⁴⁸ (MAB, corresponding to Gln¹⁶³ in -Na,K-ATPase); this bond does not alter the SjGST backbone conformation relative to SjGST alone. The three Gly residues (at positions 211, 212, and 213) in GST, together with two Pro residues at positions 216 and 217 and the Pro²²³-Arg²²⁴-Gly²²⁵-Ser²²⁶ of the linker sequence (linker positions 3-6) collectively appear to well insulate MAB from secondary structural perturbations arising in SjGST. The crystal structure of the fusion peptide thus displays two well separated and independently folded motifs, lending confidence that the structure of MAB as revealed in the fusion protein crystal is valid. Finally, it is clear from the graphical display that four MAB units are packed in each unit cell. Although theoretically the MAB conformation might be influenced by the packing force, these forces are usually quite small. Taken together, these considerations argue strongly that the conformation of MAB will not be influenced by the presence of SjGST and that carrier-mediated crystallization of active ankyrin-binding peptides fused with SjGST may represent an important and general approach to identifying the structural determinants of ankyrin binding activity in a variety of proteins.

View larger version (83K): [in this window] [in a new window]   Fig. 3.   Three-dimensional structure of the GST-MAB peptide. Top, the primary structure of the GST-MAB fusion peptide. The SjGST sequence is in red; the MAB sequence is blue. Linker sequences joining MAB to SjGST are lowercase. Bottom, the three-dimensional structure of GST-MAB, refined to an R-factor of 19.8 and Rfree of 36.5 (1% of total data) at 2.6 Å resolution, reveals two well separated and folded domains. The ankyrin-binding domain of -Na,K-ATPase is a 7-residue loop positioned on a stalk composed of anti-parallel -strands. The overall structure of SjGST is not significantly altered by the presence of the fusion construct, because superposition of SjGST onto that of SjGST-MAB yields a RMS deviation of 1.02 Å for backbone residues 1-210. The crystallographic coordinates of GST-MAB are available from the Brookhaven Protein Data Bank as accession number PDB1bie.

The Loop-on-a-Stalk Structure of MAB-- Detailed analysis of the ankyrin-binding domain within SjGST-MAB reveals an antiparallel double -strand flanking a loop composed of the seven residues MESFKNM (residues 152-158 of -Na,K-ATPase) (Fig. 4). The overall structure of MAB is suggestive of a loop on a stalk. This loop is amphipathic, presenting a hydrophobic face composed of two methionines and one phenylalanine and a hydrophilic face composed of Glu-Ser and Lys-Asn on the opposite side. A dipolar interaction between Ser¹⁴⁹ (position 234 in the crystal) and Gln¹⁶³ (crystal position 249) of the antiparallel -strands stabilizes the stalk. Presumably, in intact -Na,K-ATPase, flanking sequences would further stabilize the stalk and probably alter the positioning of the terminal residues in MAB. A search of nucleotide and protein sequence banks (GenBank^TM, Swiss-Prot) revealed exceptional conservation of MAB across species and between isoforms of -Na,K-ATPase (Fig. 5). Sequences partially homologous to MAB also exist in gastric H,K-ATPase (42), which associates with ankyrin in gastric parietal cells (43). No homologous sequences were noted in other well documented ankyrin-binding proteins (including the erythrocyte anion exchanger, the amiloride-sensitive sodium channel, the voltage-sensitive sodium channel, the Na⁺/Ca²⁺ exchanger, CD44, neurofascin, and IP3-R).

View larger version (116K): [in this window] [in a new window]   Fig. 4.   Three-axis views of the structure of the minimal ankyrin-binding domain of -Na,K-ATPase. The basic structural motif is that of a 7-residue loop on a stalk composed of antiparallel -strands. A, C, and E, surface accessibility depictions showing the amphipathic surfaces formed by the loop. B, D, and F, ribbon diagrams demonstrating the backbone contour. It is envisioned that the 7-residue loop interacts with the -hairpin tips of one or more ankyrin repeat units (see Fig. 5). The arrow in C marks the depth of the structural plane shown in E and F.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   MAB is an ATPase specific sequence that may interact with one or more ankyrin repeat units. A, sequence alignment of the minimal ankyrin-binding domain (peptide IIA) with all other proteins in GenBankTM and Swiss-Prot. Dashes represent residues identical to human Na,K-ATPase 1 subunit, residues 142-166. This sequence is well conserved among all known Na,K-ATPase  subunits and is partially conserved (68% identity) in only one other documented ankyrin-binding protein (gastric H/K-ATPase). There is no homology to the ankyrin-binding sequences in CD44, IP3-R, neurofascin, or p53bp2. However, like MAB, each of the other reported ankyrin-binding sequences are short peptides. B, hypothetical model of how MAB may interact with ankyrin, drawn to scale. Each ankyrin repeat is composed of two -helices and a -hairpin loop (5). Multiple repeat units create a structure in which helix-helix interactions form a central core, whereas the tips of the exposed -hairpin turns provide potential interprotein interaction surfaces. We envision that the 7-residue loop within MAB interacts with site(s) in ankyrin created by the tips of one or more of its -hairpin turns. Because a multiplicity of potential binding pockets would be created by the 13-24 repeat units characteristic of most ankyrins, specific and unique binding sites probably also exist for other short peptide sequences such as those that bestow ankyrin binding activity on other proteins. To effect this binding, we hypothesize that these peptides will assume a loop on a stalk structure similar to that reported here for -Na,K-ATPase.